The transmembrane chemokine CXCL16 is expressed by dendritic and vascular cells and mediates chemotaxis and adhesion of activated T cells via the chemokine receptor CXCR6/Bonzo. Here we describe the expression and shedding of this chemokine by glioma cells in situ and in vitro. By quantitative RT‐PCR and immunohistochemistry, we show that CXCL16 is highly expressed in human gliomas, while expression in normal brain is low and mainly restricted to brain vascular endothelial cells. In cultivated human glioma cells as well as in activated mouse astroglial cells, CXCL16 mRNA and protein is constitutively expressed and further up‐regulated by tumour necrosis factor α (TNFα) and interferon‐γ (IFNγ). CXCL16 is continuously released from glial cells by proteolytic cleavage which is rapidly enhanced by stimulation with phorbol‐12‐myristate‐13‐acetate (PMA). As shown by inhibitor studies, two distinct members of the disintegrin‐like metalloproteinase family ADAM10 and 17 are involved in the constitutive and PMA‐induced shedding of glial CXCL16. In addition to the chemokine, its receptor CXCR6 could be detected by quantitative RT‐PCR in human glioma tissue, cultivated murine astrocytes and at a lower level in microglial cells. Functionally, recombinant soluble CXCL16 enhanced proliferation of CXCR6‐positive murine astroglial and microglial cells. Thus, the transmembrane chemokine CXCL16 is expressed in the brain by malignant and inflamed astroglial cells, shed to a soluble form and targets not only activated T cells but also glial cells themselves.